To avoid spleen-dependent killing mechanisms parasite-infected erythrocytes (IE) of Plasmodium falciparum malaria patients have the capacity to bind to endothelial receptors. This binding also known as sequestration, is mediated by parasite proteins, which are targeted to the erythrocyte surface. Candidate proteins are those encoded by P. falciparum multicopy gene families, such as var, rif, stevor or PfMC-2TM. However, a direct in vivo proof of IE sequestration and expression of multicopy gene families is still lacking. Here, we report on the analysis of IE from a black African immigrant, who received the diagnosis of a malignant lymphoproliferative disorder and subsequently underwent splenectomy. Three weeks after surgery, the patient experienced clinical falciparum malaria with high parasitemia and circulating developmental parasite stages usually sequestered to the vascular endothelium such as late trophozoites, schizonts or immature gametocytes. Initially, when isolated from the patient, the infected erythrocytes were incapable to bind to various endothelial receptors in vitro. Moreover, the parasites failed to express the multicopy gene families var, A-type rif and stevor but expression of B-type rif and PfMC-2TM genes were detected. In the course of in vitro cultivation, the parasites started to express all investigated multicopy gene families and concomitantly developed the ability to adhere to endothelial receptors such as CD36 and ICAM-1, respectively. This case strongly supports the hypothesis that parasite surface proteins such as PfEMP1, A-type RIFIN or STEVOR are involved in interactions of infected erythrocytes with endothelial receptors mediating sequestration of mature asexual and immature sexual stages of P. falciparum. In contrast, multicopy gene families coding for B-type RIFIN and PfMC-2TM proteins may not be involved in sequestration, as these genes were transcribed in infected but not sequestered erythrocytes.

Malaria claims about half a million lives each year. Plasmodium falciparum , the causative agent of the most severe form of the disease, uses proteins that are translocated to the surface of infected erythrocytes for immune evasion. To circumvent the detection of these gene products by the immune system, the parasite evolved a complex strategy that includes gene duplications and elaborate sequence polymorphism. STEVORs are one family of these variant surface antigens and are encoded by about 40 genes. Using deep RNA sequencing of blood-stage parasites, including free merozoites, we first established stevor expression of the cultured isolate and compared it with published transcriptomes. We reveal a biphasic expression of most stevor genes and confirm this for individual STEVORs at the protein level. The membrane topology of a rhoptry-associated variant was experimentally elucidated and linked to host cell invasion, underlining the importance of this multifunctional protein family for parasite proliferation.

Variant surface antigens (VSA) exposed on the membrane of Plasmodium falciparum infected erythrocytes mediate immune evasion and are important pathogenicity factors in malaria disease. In addition to the well-studied PfEMP1, the small VSA families RIFIN, STEVOR and PfMC-2TM are assumed to play a role in this process. This study presents a detailed comparative characterization of the localization, membrane topology and extraction profile across the life cycle of various members of these protein families employing confocal microscopy, immunoelectron microscopy and immunoblots. The presented data reveal a clear association of variants of the RIFIN, STEVOR and PfMC-2TM proteins with the host cell membrane and topological studies indicate that the semi-conserved N-terminal region of RIFINs and some STEVOR proteins is exposed at the erythrocyte surface. At the Maurer’s clefts, the semi-conserved N-terminal region as well as the variable stretch of RIFINs appears to point to the lumen away from the erythrocyte cytoplasm. These results challenge the previously proposed two transmembrane topology model for the RIFIN and STEVOR protein families and suggest that only one hydrophobic region spans the membrane. In contrast, PfMC-2TM proteins indeed seem to be anchored by two hydrophobic stretches in the host cell membrane exposing just a few, variable amino acids at the surface of the host cell. Together, the host cell surface exposure and topology of RIFIN and STEVOR proteins suggests members of these protein families may indeed be involved in immune evasion of the infected erythrocyte, whereas members of the PfMC-2TM family seem to bear different functions in parasite biology.

Virulence of the most deadly malaria parasite Plasmodium falciparum is linked to the variant surface antigen PfEMP1, which is encoded by about 60 var genes per parasite genome. Although the expression of particular variants has been associated with different clinical outcomes, little is known about var gene expression at the onset of infection. By analyzing controlled human malaria infections via quantitative real-time PCR, we show that parasite populations from 18 volunteers expressed virtually identical transcript patterns that were dominated by the subtelomeric var gene group B and, to a lesser extent, group A. Furthermore, major changes in composition and frequency of var gene transcripts were detected between the parental parasite culture that was used to infect mosquitoes and Plasmodia recovered from infected volunteers, suggesting that P. falciparum resets its var gene expression during mosquito passage and starts with the broad expression of a specific subset of var genes when entering the human blood phase. Parasites of the species Plasmodium falciparum, which are responsible for the most severe forms of malaria, escape from the human immune response by antigenic variation. A repertoire of 60 var genes codes for a broad range of different variant antigens presented on the surface of infected erythrocytes. These antigens mediate adhesion to blood vessels, thereby disturbing the blood microcirculation and causing life-threatening organ dysfunctions. To better understand antigenic variation in vivo we analyzed the var gene expression profiles in blood samples from 18 malaria naïve volunteers, who were experimentally infected with cryopreserved sporozoites isolated from Anopheles mosquitoes. Our in-depth analysis revealed a broad, but remarkably uniform expression pattern of a specific set of var gene variants. Moreover, the results clearly show that this var gene expression program is specifically activated after mosquito transmission of the parasite. These findings are of particular importance for our understanding of the strategy of malaria parasites to establish and maintain infections in the human host.

Several essential biochemical processes are situated in mitochondria. The metabolic transformation of mitochondria in distinct lineages of eukaryotes created proteomes ranging from thousands of proteins to what appear to be a much simpler scenario. In the case of Entamoeba histolytica, tiny mitochondria known as mitosomes have undergone extreme reduction. Only recently a single complete metabolic pathway of sulfate activation has been identified in these organelles. The E. histolytica mitosomes do not produce ATP needed for the sulfate activation pathway and for three molecular chaperones, Cpn60, Cpn10 and mtHsp70. The already characterized ADP/ATP carrier would thus be essential to provide cytosolic ATP for these processes, but how the equilibrium of inorganic phosphate could be maintained was unknown. Finally, how the mitosomal proteins are translocated to the mitosomes had remained unclear. We used a hidden Markov model (HMM) based search of the E. histolytica genome sequence to discover candidate (i) mitosomal phosphate carrier complementing the activity of the ADP/ATP carrier and (ii) membrane-located components of the protein import machinery that includes the outer membrane translocation channel Tom40 and membrane assembly protein Sam50. Using in vitro and in vivo systems we show that E. histolytica contains a minimalist set up of the core import components in order to accommodate a handful of mitosomal proteins. The anaerobic and parasitic lifestyle of E. histolytica has produced one of the simplest known mitochondrial compartments of all eukaryotes. Comparisons with mitochondria of another amoeba, Dictystelium discoideum, emphasize just how dramatic the reduction of the protein import apparatus was after the loss of archetypal mitochondrial functions in the mitosomes of E. histolytica. All eukaryotic organisms have mitochondria, organelles cordoned by a double membrane, which are descendants of an ancestral bacterial endosymbiont. Nowadays, mitochondria are fully integrated into the context of diverse cellular processes and serve in providing energy, iron-containing prosthetic groups and some of the cellular building blocks like lipids and amino acids. In multi-cellular organisms, mitochondria play an additional vital role in cell signaling pathways and programmed cell death. In some unicellular eukaryotes which inhabit oxygen poor environments, intriguing mitochondrial adaptations have taken place resulting in the creation of specialized compartments known as mitosomes and hydrogenosomes. Several important human pathogens like Entamoeba histolytica, Giardia intestinalis, Trichomonas vaginalis and microsporidia contain these organelles and in many cases the function and biogenesis of these organelles remain unknown. In this paper, we investigated the protein import pathways into the mitosomes of E. histolytica, which represent one of the simplest mitochondria-related compartment discovered yet. In accordance with the limited organellar proteome, we show that only core components of mitochondria-related protein import machines are present in E. histolytica to serve for the import of a small set of substrate proteins.

Deformability of Plasmodium falciparum gametocyte-infected erythrocytes (GIE) allow them to persist for several days in blood circulation and to ensure transmission to mosquitoes. Here, we investigate the mechanism by which the parasite proteins STEVOR exert changes on GIE deformability. Using the microsphiltration method, immunoprecipitation and mass spectrometry, we produce evidence that GIE stiffness is dependent on the cytoplasmic domain of STEVOR that interacts with ankyrin complex at the erythrocyte skeleton. Moreover, we show that GIE deformability is regulated by PKA-mediated phosphorylation of STEVOR C-terminal domain at a specific serine residue (S₃₂₄). Finally, we show that the increase of GIE stiffness induced by sildenafil (Viagra®) is dependent on STEVOR phosphorylation status and on another independent mechanism. These data provide new insights into mechanisms by which phosphodiesterases inhibitors may block malaria parasite transmission.